to 96-well plates and alkaline phosphataseconjugated PGE2 antibodies were added to the sample wells. The samples were incubated at room temperature for 2 h. Sample wells were washed, and p-nitrophenyl phosphate substrate solution was added. Finally, the samples were incubated at room temperature for 1 h, and absorbance was read according to the manufacturer’s instructions. Statistical Analysis All data are expressed as mean 6 SD. Differences between the control and treatment group were evaluated by Student’s t-test using Statview software. A P,0.01 was considered statistically significant. Results UV-absorbing Properties and Photosafety of Afzelin Inflammatory Cytokine Assay The cells were irradiated with the indicated UVB doses and then incubated with the indicated afzelin concentration for 12 h. After 12 h, TNF-a and IL-6 concentrations in the culture supernatant were measured using ELISA kits, according to the manufacturer’s instructions. Briefly, culture supernatants were added to 96-well plates, and diluted biotinylated TNF-a or IL-6 was added to the sample wells. The samples were incubated at room temperature for 3 h, after which the wells were washed. Streptavidin-HRP was distributed to the sample wells and the plate was incubated for 30 min at room temperature. The wells were washed, and 3, 39, 5, 59-tetramethylbenzidine substrate Effects of Afzelin on UVB-Induced Cell Damage 10 Effects of Afzelin on UVB-Induced Cell Damage measured by staining HaCaT cells with JC-1 followed by fluorescence microscopy analysis. 8941386 HaCaT cells were incubated with the JC-1 probe for 30 min. Mean fluorescence, expressed as a percentage of the control, indicates the ratio of high/low mitochondrial membrane potential. Representative data, n = 
3. C. DHR 123 was employed to detect mitochondrial hydrogen peroxide. Reactive oxygen species -induced DHR 123 fluorescence was measured using a spectrophotometer. D. Enzyme-linked immunosorbent assay analysis of cytochrome c in cytosolic and mitochondrial fractions of HaCaT cells exposed to UVB. HaCaT cells were treated with afzelin for 12 h after exposure to UVB radiation. Then, the cells were processed for cytochrome c analysis. Data are means 6 SD, n = 3. 1P,0.01 compared with the vehicle-treated group, “P,0.01 compared with the UVB -treated group. doi:10.1371/journal.pone.0061971.g006 and non-phototoxic molecules . Thus, afzelin was considered a non-phototoxic compound. Afzelin Suppresses the Increase in Intracellular ROS Induced by UVB Radiation Until now, we found that UV-induced DNA damage was reduced by UV-absrobing activity of afzelin. As a next step, we investigated cellular activities of afzelin except UV-absorbing activity. To this end, afzelin was treated after UV irradiation to exclude the UV-absorbing properties of afzelin. Intracellular ROS generation was monitored to investigate the effects of afzelin on UVB-induced oxidative stress in HaCaT cells. Afzelin was added to the 7751958 cells after UVB irradiation. The antioxidative activity of afzelin was determined with the oxidant-sensitive fluorescent dye DCFH-DA in UVB-irradiated HaCaT cells. The UVB-induced increase in ROS generation decreased significantly in a concentration-dependent manner following afzelin treatment. Fluorescence microscopy showed that 100 mg/ml afzelin Elesclomol completely inhibited formation of the DCF green fluorescent dye. A 20 mJ/cm2 UVB dose was used in the ROS experiments. No effect on cell viability of afzelin was observed either witho